The continuous production of blood cells is dependent on the proper functioning of hematopoietic stem cells (HSC). HSC have the unique capacity to self-renew and to give rise to all cells of the hematopoietic lineage for the lifetime of an organism. Signal-transduction pathways integrate critical stimuli HSC receive that produce unique biological outcomes. These pathways are required for physiological function and when disrupted can cause hematological diseases. The mTOR pathway responds to nutrients, growth factors, and cellular energy levels, and has recently been implicated in regulating HSC function. The overarching goal of this proposal is to discern the function of the mTOR complexes (mTORCs) in adult HSC and hematopoiesis. Key mTORC regulatory genes will be conditonally deleted in adult mice (Raptor for mTORC1, and Rictor for mTORC2). Furthermore, mechanistic insight into how the mTORCs influences hematopoietic differentiation decisions, via the use of advanced intracellular flow-cytometry techniques, single-cell clonogenic assays, gene-expression analysis, and compound mouse crosses will be acquired. The Specific Aims are: 1) To precisely discern the role of mTorc1 in HSC function using mice with an inducible "knock- out" allele of Raptor. 2) To define the function of Raptor (mTorc1) in lineage-choice decisions and myeloid cell differentiation. 3) To determine the contribution of Rictor (mTorc2) in abnormal hematopoiesis evoked by Raptor loss. Dr. Demetrios Kalaitzidis, a research fellow in the Department of Hematology/Oncology at Children's Hospital Boston (CHB), has outlined a 5-year career plan that has built upon his past background and experience as a researcher in hematopoietic signal-transduction pathways. Under the mentorship of Dr. Scott Armstrong, a recognized leader in the fields of stem-cell biology, gene-expression analysis, and leukemia, he will utilize mouse models to dissect the mTOR pathway in adult hematopoiesis. Together they have crafted a career- development plan to further Dr. Kalaitzidis'scientific skills through technical training, course-work, seminar and conference attendance, and the organization of a scientific-advisory committee composed of preeminent researchers in the fields of hematopoiesis and signal transduction. The Department of Hematology/Oncology at CHB, with its world-class resources and critical mass of researchers, is ideally suited for this career- development proposal to be executed. PUBLIC HEALTH RELEVANCE: The lifelong requirement for blood-cell production is critically dependent on the proper functioning of blood (hematopoietic) stem cells. The unique properties of these stem cells are dependent on signaling pathways that respond to and integrate a number of internal and external factors, including nutrients and growth factors. When these pathways are altered, they can lead to blood disorders and diseases. Through the use of advanced gene-targeting technologies in mice, the proposed work will delineate the role of one of these pathways (mTOR) implicated in adult blood-stem cell and blood-system function. The proposed work will advance our knowledge of the blood system and lead to the development of novel therapeutic interventions for blood disorders.